Bacillus cereus strain AS4-12

ABSTRACT

A novel strain of Bacillus cereus, designated AS4-12, has been isolated from the environment. The strain AS4-12 is one of a number of B. cereus strains which are useful as biocontrol agents to combat fungal damping off disease in field crop plants, and strain AS4-12 has exhibited the best performance among a large number of natural isolates in fostering the emergence and growth of alfalfa plants under normal field conditions in the upper midwestern U.S.

TECHNICAL FIELD

The present invention is in the general field of bacteriology andrelates, in particular, to a novel strain of bacteria useful as abiocontrol agent in field applications.

BACKGROUND OF INVENTION

Significant research has been conducted in recent years on the use ofbiological agents to increase agricultural productivity and efficiency.Biological control based on the use of microorganisms to suppress plantpests or supplement plant growth offers an attractive alternative tochemical pesticides which are less favored than they have previouslybeen because of concerns about human health and environmental quality.Several screening programs have been used before to isolate biologicalagents which are effective in the laboratory or in the field to combatpests or facilitate plant growth.

An example of a biological control agent into which significantscientific and economic development has occurred is the use of theBacillus thuringiensis. It was recognized that B. thuringiensis strainsproduced toxic proteins which have the ability to specifically killcertain insects and that initial inquiry led to a significant researchwhich has proceeded to identify a large number of B. thuringiensisstrains having variations and target range in efficacy. In addition,research has been conducted on methods for stabilizing and applying suchtoxins, or strains harboring them, to a wide variety of field cropsituations. It was also discovered that knowledge of B. thuringiensisstrains was largely transferable to new strains since the toxinsrequired for biological control and methods for preparing inocula foruse in the field were generally similar among strains.

Previously it has been found that a specific strain of Bacillus cereus,which has been referred to both as UW85 and by its ATCC designation53522 has biocontrol efficacy in many applications. The UW85 B. cereusstrain was found to protect alfalfa seedlings from damping off caused byPhytophthora medicaginis, tobacco seedlings from Phytophthora nicotianaecucumber fruits from rot caused by Pythium aphanidermatum, and peanutsfrom Sclerotinia minor. UW85 is also described, by reference to its ATCCnumber in U.S. Pat. No. 4,877,738. It was later found that UW85 producedtwo antifungal compounds which contribute independently to itssuppression of damping off fungi due to antifungal and antibacterialactivity. The more potent of these compounds, a novel aminopolyol hasbeen designated zwittermicin A while the second compound, not wellcharacterized, has been provisionally designated antibiotic B.

"Biological control" is defined as pathogen suppression by the use of asecond organism. Mechanisms of biological control are diverse. Forexample, certain enteric bacteria have been examined for theirusefulness in biological control of root rot in alfalfa. It is believedthat control is obtained by competition between the enteric bacteria andthe fungi for space on the surface of the alfalfa roots. In contrast, atoxin produced by one species of bacteria may be used to control anotherspecies of bacteria that appears as a pathogen. Bacterially producedantibiotics are an example of such toxins. The toxin can be isolatedfrom the species producing it and administered directly, as is thecommon procedure with penicillin, or the species itself may beadministered under appropriate circumstances to produce the toxin insitu. Once identified, such toxins produced by soil-dwelling bacteriamay have utility in diverse other areas as antifungal or antibioticagents.

SUMMARY OF THE INVENTION

The present invention is summarized in that a novel Bacillus cereusstrain, here designated AS4-12, ATCC No. 55609, has been isolated fromthe environment. Strain AS4-12 has been found to have increased efficacyin fostering the growth and establishment of alfalfa plants in the fieldenvironment of the upper mid-western United States.

The present invention is further characterized in that a method isdescribed to foster the growth of alfalfa stands by the application ofan inoculum including as its active agents a novel Bacillus cereusisolate designated AS4-12, ATCC No. 55609.

Other objects, advantages, and features of the present invention willbecome apparent from the following specification.

DETAILED DESCRIPTION OF THE INVENTION

An original bacterial strain, isolated from soil, exerts biologicalcontrol over species of fungi responsible for damping off and root rotin plants. The strain has been deposited in the American Type CultureCollection, given the designation ATCC 55609, and shall hereinafter bereferred to either as Strain AS4-12 or ATCC 55609.

It is further anticipated that certain mutants of AS4-12 also providebiological control comparable to that provided by AS4-12. Thesebacterial strains may also be obtained in substantially pure cultures. A"substantially pure" culture shall be deemed a culture of a bacteria,containing no other bacterial species in quantities sufficient tointerfere with replication of the culture or to be detectable by normalbacteriological techniques. In addition, it has been discovered that thebiological control is exerted principally by means of one or more toxinsproduced by the bacterial strain.

Strain AS4-12 is one of a group of Bacillus cereus strains that areuseful biocontrol agents due, at least in part, to the fact that theynaturally synthesize antibiotic agents, notably an antibiotic which isthe subject of a co-pending patent application. The antibiotic or toxinis found in supernatant fluid and other bacteria-free fluid and culturemedium removed from a culture of AS4-12 or of its protecting mutants,has been found to be a "protecting toxin," as that term is definedbelow. This toxin has been so characterized as to be identifiableindependent of its source in cultures of Bacillus cereus, and is knownand by the coined term "zwittermicin A." Another fraction from thesupernatant fluid from a culture of B. cereus ATCC 53522 has been foundbiologically active, having a zoolysin capability to zoospores ofPhytophthra medicaginis (Pmm), but, as revealed below, this zoolysinactive fraction does not have the antifungal activity of the antibiotic.Bacillus cereus antibiotic zwittermicin A has been found to be a highlywater soluble molecule of about 396 daltons. The molecule is azwitterion, that is it contains both acid and base groups, includes twoamino groups, and is a poly-alcohol.

The method by which the biological control referred to in the precedingparagraph may be verified to exist is the "plant protection assay"detailed below. "Biological control" of fungi causing damping off androot rot shall be deemed to exist if, when an effective quantity ofAS4-12, its mutants that exhibit biological control, the anti-fungaltoxin produced by them, or any other compound or molecule is placed inthe soil or other growing medium in the immediate vicinity of the plantto be protected, a statistically significant reduction in the symptomsof damping off or root rot occurs in the presence of one or morepathogens causing these diseases. An "effective quantity" to combatdamping off and root rot shall be that quantity sufficient to result insuch a visibly significant reduction of symptoms. Clearly, if noquantity of a bacteria or any toxin or other compound is an effectivequantity as so defined, that bacteria, toxin, or compound is not capableof exerting biological control over the fungi causing damping off androot rot.

AS4-12 and those of its mutants capable of exerting such biologicalcontrol shall sometimes be referred to collectively as "protecting"bacteria. Bacillus cereus antibiotic and other toxins capable ofexerting such biological control shall sometimes be referred to as"protecting" compounds or toxins. Plants, including seeds, seedlings,and mature plants, treated with such an effective quantity of protectingbacteria, their toxins, or Bacillus cereus antibiotic shall be referredto as "protected" from root rot or damping off.

The following is a disclosure of the plant protection assay whereby atest material such as a bacteria, a toxin, or the like, may be testedfor its ability to exert biological control over a fungus capable ofcausing the symptoms of damping off or root rot. The seed or seedling ofthe plant to be protected is planted in a planting medium in thepresence of damping off or root rot causing fungi. The planting mediummay be a damp soil containing such fungi, vermiculite in water with thefungi present either in the vermiculite and water or in or on the seedor seedling, an agar-based formulation, or any other planting medium inwhich the seed or seedling will grow and the fungi may freely develop.The bacteria, toxin, or other test material is placed at least in theimmediate vicinity of the seed or seedling. Such placement shall beunderstood to be in the "immediate vicinity" of the seed or seedling ifany soluble test material or any soluble exudate of a bacteria beingtested will be in actual contact with the germinating seedling.

Preferably, if seed is used, the seed is coated with the test material,and when the test material is so used with respect to a seed, it shallbe referred to hereinafter as a "seed inoculum." The process of coatingseed with a seed inoculum is generally well known to those skilled inthe art, and any conventional method that does not require conditionssufficiently harsh to kill bacteria, to harm the seeds, or to destroytoxins or other materials included in the seed inoculum, is adequate. Aneasy and preferred method is to suspend or dissolve the test material ina 1.5% aqueous solution of methyl cellulose. For convenience, it will bepresumed hereinafter that the seed inoculum is a bacteria suspended inthe methyl cellulose, although a dissolvable material such as abacterial toxin may be handled in the same manner or in a differentmanner to the same effect. The plant seed to be protected is added tothe suspension and is mixed vigorously with it to coat the surface ofthe seed with the suspension. The seed may then be dried, preferably bybeing placed within a laminar flow hood on a sterile surface such as asterile petri plate. The result is a dry, seed inoculum-coated seed.When the coated seed is planted in the planting medium, the testmaterial accompanies it to reside in the immediate vicinity of the seed.

After a time sufficient for seedling growth and the expression of thesymptoms of damping off, seedlings developing from the planted seed maybe evaluated for visual evidence of protection, when compared tocontrols. In strains of alfalfa, soybeans, and snap beans known to bevulnerable to damping off, one to two weeks of growing time in a growthchamber at 24° C. with a 12 hour photoperiod was found o be a periodsufficient for the expression of symptoms of damping off when seedlingswere being grown in test tubes containing roughly 10³ zoospores of Pmmor comparable, damping off-causing fungi. Protected seeds developed intoseedlings visually indistinguishable from uninfected seeds while controlseedlings developing from unprotected seeds were killed or, in the caseof snap beans, exhibited brown lesions on roots and stems, stuntedroots, rotted roots, and other visually apparent symptoms of root rot.

Protecting mutants of AS4-12 include both naturally occurring andartificially induced mutants. For example, AS4-12 is generally sensitiveto the antibiotics rifampicin and neomycin. However, it is expected thatnaturally occurring mutants of AS4-12 can be isolated that exhibitedresistance to one or the other of these antibiotics. Certain of thesemutants, as well as one naturally occurring mutant distinguishable fromthe parent AS4-12 strain by the appearance of its colonies, will befound to protect alfalfa plants in the plant protection assay. Othermutants of AS4-12 can be artificially induced by subjecting AS4-12 tothe mutagen N-methyl-nitrogoguanidine in conventional ways. Similarmutants have been made from other useful B. cereus strains, such as UW85(ATCC 53522), as described in U.S. Pat. No. 4,877,738, the disclosurewhich is hereby incorporated by reference.

In terms of conventional bacteriological morphology and colony analysistechniques, strain AS4-12, ATCC 55609, is indistinguishable from UW85,ATCC 53522. The differences between the strains largely relate to thestatistical difference in actual field performance conditions, asreported below. In essence, strain AS4-12 provides a statisticallysignificant higher level of pathogen suppression than does UW85.

EXAMPLE 1 Origin of Strains

The geographic origins, and physical and chemical characteristics of thesoil samples used in this study are listed in Table 1. All samples weretaken from the surface horizon. Measurements of soil pH, organic matter,and the particle size were made by the University of Wisconsin Soil &Plant Analysis Laboratory (Madison, Wis.).

                                      TABLE 1                                     __________________________________________________________________________    Characteristics of soils used in this study                                                            % Sand, Silt,                                                                        Most recent                                   Soil/Site                                                                              Country                                                                              pH % Organic.sup.a                                                                     Clay.sup.b                                                                           vegetation                                    __________________________________________________________________________    Lutz     Panama  nd.sup.c                                                                        5.5   nd     Forest                                        Snyder-Molino                                                                          Panama nd 6.2   nd     Forest                                        Barbour-Lathrop                                                                        Panama nd 7.1   nd     Forest                                        Moroceli Honduras                                                                             nd 1.7   nd     Maize-Beans                                   San Matias                                                                             Honduras                                                                             5.8                                                                              1.5   nd     Maize-Beans                                   LaVega1  Honduras                                                                             6.3                                                                              2.3   nd     Beans                                         LaVega5  Honduras                                                                             5.9                                                                              2.5   nd     Sorghum-Maize                                 Arlington WI                                                                           USA    6.9                                                                              4.2   23, 68, 9                                                                            Alfalfa                                       Hancock WI                                                                             USA    6.6                                                                              0.8   87, 8, 5                                                                             Alfalfa-Oats                                  Marshfield WI                                                                          USA    6.5                                                                              3.1   23, 72, 5                                                                            Alfalfa                                       Lancaster WI                                                                           USA    7.1                                                                              2.3   19, 68, 13                                                                           Alfalfa                                       Madison WI                                                                             USA    7.0                                                                              3.2   27, 56, 17                                                                           Alfalfa                                       Taos NM  USA    8.1                                                                              4.3   nd     Pasture                                       Tifton GA                                                                              USA    6.3                                                                              1.0   87, 12, 1                                                                            Tobacco                                       Douglas Gully                                                                          Australia                                                                            6.1                                                                              2.2   nd     Vineyard                                      Lelystad Netherlands                                                                          7.5                                                                              1.8   51, 36, 13                                                                           Potatoes                                      __________________________________________________________________________

Isolation and identification of B. cereus isolates

The bacterial strains and isolates and their origins are listed in Table2. Bacteria collected from soybean roots were isolated as previouslydescribed from plants grown in a field plot in Madison, Wis. (Table 2).The 10 remaining bacteria collected in this study were isolated byplacing either an entire alfalfa root or 1 g of soil in a test tubecontaining 9 ml water and sonicating it for 30 sec. in a Model 2200 bathsonicator (Branson Ultrasonics Corp., Danbury, Conn.), Serial 10-folddilutions of the suspensions were made in water, and 0.1 ml fromdilutions ranging from 10⁻¹ to 10⁻⁵ were spread on either 1/10 strengthtrypticase soy agar (1/10-strength TSA) (Becton Dickinson MicrobiologySystems, Cockeysville Md.) or MinIC media. Plates were incubated at roomtemperature or 28° C. for one to three days. The plates containingisolated colonies were used for further study. Colonies that had themorphology typical of B. cereus (flat, broad, and cream colored) werepicked and streaked for isolated colonies. As a partial selection for B.cereus, during either the initial plating or subsequent streak plating,the medium was supplemented with polymyxin (25 μg/ml), cycloheximide(100 μg/ml), and ampicillin (50 μg/ml). All isolates were tested forhemolysis of blood agar, which is diagnostic of B. cereus, and thosethat were non-hemolytic were removed from the collection. Blood agar wasobtained from the Wisconsin State Hygiene Lab, Madison, Wis. Isolateswere stored on 1/2-strength trypticase soy agar (1/2-strength TSA)slants. Alfalfa plants were grown from seed in soil from Arlington Wis.for 21 days in a growth chamber at 24° C. with a 12 hour photoperiod anda light intensity of 244 microeinsteins/m^(2/) s. Soybean plants weregrown from seed in a field plot in Madison Wis.

                  TABLE 2                                                         ______________________________________                                        Strains and isolates used in this study                                       Strain(s)/Isolates    Origin                                                  ______________________________________                                        ATCC7064,ATCC27877,ATCC12826                                                                        American Type                                                                 Culture Collection                                      BGSC6A3,BGSC6E1,      Bacillus Genetic                                        BGSC6E2,BGSC4A9,      Stock Center                                            BGSC4B1,BGSC4C3,                                                              HD1,BGSC4E1,                                                                  BGSC4F1,BGSC4G1,                                                              BGSC4H1,BGSC4I1,                                                              BGSC4J1,BGSC4S2                                                               T                     U.W.Bacteriology                                                              Dept. Collection                                        UW85                  Alfalfa root,                                                                 Arlington, WI (26)                                      Soy130                Soybean root, Walnut                                                          St. Farm,                                                                     Madison, WI                                             ALF1,ALF9,ALF10,      Roots of alfalfa                                        ALF13,ALF19,ALF23,    plants planted                                          ALF52,ALF53,ALF79,    in soil from                                            ALF83,ALF85,ALF94,    Arlington, WI and                                       ALF95,ALF98,ALF99,    grown in growth                                         ALF108,ALF109,        chamber                                                 ALF115,ALF117,ALF133,                                                         ALF137,ALF144,                                                                ALF154,ALF157,ALF161,                                                         ALF166,ALF167,                                                                ALF173                                                                        LUTZ21,LUTZ58,LUTZ128 Lutz soil                                               SNY14,SNY42,SNY44,SNY45,SNY73                                                                       Snyder-Molino soil                                      BAR78,BAR145,BAR177   Barbour-Lathrop soil                                    MOR1,MOR28,MOR37      Moroceli soil                                           SM32,SM43,SM44        San Matias soil                                         VGA19,VGA118,VGA137   LaVega1 soil                                            VGA562,VGA577,VGA598  LaVega5 soil                                            AS7-4,AS8-4,AG8-13,AS4-12,ARL8                                                                      Arlington soil                                          HS1-3,HS23-11,HS24-8,HS24-9                                                                         Hancock soil                                            MS1-9,MS3-2,MS8-2     Mansfield soil                                          LS2-2,LS2-12,LS33-2   Lancaster soil                                          WS4-12,WS8-8,WS10-15,WS16-4,WS22-12                                                                 Madison soil                                            TNM68,TNM155,TNM243   Taos soil                                               TG38,TG42,TG126       Tifton soil                                             DGA34,DGA37,DGA84,DGA94                                                                             Douglas Gully soil                                      LN24,LN75,LN100       Lelystad soil                                           ______________________________________                                    

Based on the profiles of fatty acids from 47 isolates analyzed by fiveStar Labs (Branford Conn.) and Microbial ID (Newark Del.), all of theisolates were classified as members of the B. cereus group, whichincludes the species B. mycoides, B. anthracis and B. thuringiensis. Theunique rhizoidal morphology of B. mycoides strains differentiates themfrom B. cereus, and none of the isolates in this collection display B.mycoides-like morphology. B. anthracis is not hemolytic and is usuallysensitive to ampicillin and therefore was probably excluded from thiscollection. Differentiation between B. cereus and B. thuringiensis isdifficult with standard methods. Therefore we have followed currentrecommendations and considered all isolates gathered in this study as B.cereus. Strains BGSC4A9, BGSC4B1, BGSC4C3, HD1, BGSC4E1, BGSC4F1,BGSC4G1, BGSC4H1, BTSC4I1, BGSC4J1 and BGSC4S2 were previouslyclassified by others as B. thuringiensis, and that species designationwas retained for those strains.

Assay for sensitivity to phage P7

The phages P7 (ATCC75237) and PB were used to help characterize thestrains. The susceptibility of B. cereus strains to infection by phageP7 has proven to have a strong co-relation to biocontrol utility andantibiotic production. To propagate these phages, we spread a mixture ofmelted soft agar (4 g agar/1) with approximately 10⁶ PFU of phage and anexcess of B. cereus strain UW85 on 1/2-strength TSA plates. Plates wereincubated overnight at 28° C. and then the soft agar was scraped off theplates and suspended in 1/2-strength trypticase soy broth (1/2-strengthTSB), (1 ml/plate). Agar and cells were removed by centrifugation, andthe supernatant solution was passed through a 0.2 μm filter. Phagetiters were typically 1×10¹⁰ PFU/ml.

To screen large numbers of isolates for P7 sensitivity, grids of 48isolates were grown on 1/10-strength TSA and then cells were transferredwith a metal replicator onto 1/10-strength TSA plates that had beenspread with dilutions of P7 such that they contained approximately 10⁸,10⁴, and 10³ PFU/plate. A 1/10-strength TSA plate containing no phagewas used as a control. Isolates that appeared to form patches withdecreased growth or plaques on plates containing P7 were tested in thesoft-agar overlay assay (described below) to determine if they wereP7^(s). Most isolates that were P7^(r) in the primary screen were notre-tested.

In the second test for sensitivity of bacterial isolates to P7, eachisolate was grown on 1/2-strength TSA and cells were scraped off platesand mixed in soft agar overlays to form lawns on fresh 1/2-strength TSAplates. Ten-fold dilutions of P7 were placed in 5-μl drops on theplates, which were then incubated at 28° C. If plaques appeared, thestrain was designated P7-sensitive (P7^(s)). Lawns of two isolates, ARL8and HS23-11, were cleared by undiluted drops of P7, but P7 did not formisolated plaques on these isolates at lower concentrations. The clearingdue to high titer drops appeared to be due to P7 rather than a chemicalpresent in UW85 lysates, since high titer drops of lysates of PB, whichproduces turbid plaques on UW85, did not cause clearing on lawns of ARL8and HS23-11. Therefore these strains were also scored P7^(s). Isolateswhose lawns appeared unaffected by P7 were scored P7^(r).

Assay for inhibition of Erwinia herbicola

Inhibition of E. herbicola LS005 was assayed as described in Silo-Suh etal. Appl. Environ. Microbiol., 60:2023-2030 (1994), with the followingmodifications. Three-day-old cultures of each B. cereus isolate grown in1/2-strength TSB were tested to determine whether they inhibited E.herbicola on 1/1000-strength TSA plates. Isolates that produced visiblezones of inhibition of E. herbicola were tested again. Isolates thatproduced visible zones of inhibition in both tests were scored Eh⁺.Isolates that did not noticeably inhibit E. herbicola in each of twoinitial tests were scored Eh⁻. Some B. cereus isolates did not inhibitE. herbicola during initial testing but did after storage at -20° C.,and certain isolates (ALF115,HD1 and BGSC4S2) had variable phenotypesproducing either small zones of inhibition or no zone in subsequenttests; these were classified Eh⁻.

Assay for zwittermicin A and antibiotic B

Zwittermicin A and antibiotic B were identified in culture supernatantsby cation exchange chromatography using CM SEP-PAK cartridges(Millipore, Millford, Mass.) followed by high voltage paperelectrophoresis (HVPE). The cation fraction from the equivalent of 4 mlof culture supernatant was applied to the paper, which was stained withsilver nitrate after electrophoresis, described in Silo-Suh et al.supra. Isolates that produced material indistinguishable from eitherauthentic zwittermicin A or authentic antibiotic B in HVPE weredesignated zwittermicin A producers or antibiotic B producers,respectively. To verify the structural identity of zwittermicin Aproduced by nine representatives of the collection of isolates,putative-zwittermicin A was purified from these isolates, and subjectedto proton nuclear magnetic resonance Spectroscopy (¹ H-NMR) and fastatom bombardment mass spectrometry.

Assay for suppression of alfalfa damping-off

Bacterial isolates were grown for three days in 1/2-strength TSB andtested in an assay for damping-off. Each isolate was tested on fifteenplants in each of 7 separate experiments with the following exceptions:strain ATCC12826 was omitted from experiments 1 and 2; strain BAR78 wasomitted from experiment 4; and strains WXS8-8 and LS2-12 were omittedfrom experiment 5. Statistical analyses (analysis of variance, Dunnet'scomparison test, standard error of least squared mean) were conductedusing the SAS Computer Program (SAS Institute, Raleigh, N.C.). The datafrom the seven experiments were pooled and analyzed as a singleexperiment with seven blocks.

Testing diversity of strains

To estimate the diversity of zwittermicin A and antibiotic B-producers,we sought to determine the minimum number of unique zwittermicin Aand/or antibiotic B-producing strains in our collection. We consideredisolates to be distinct strains only if phenotypic differences betweenthem could be shown. Therefore, isolates were subjected to a series ofphenotypic tests. All characterization was performed on isolates thathad been colony purified on 1/2-strength TSA. To test for antibioticresistance, isolates were streaked on 1/2-strength TSA containingtetracycline (5 μg/ml), neomycin (5 μg/ml), or chloramphenicol (1μg/ml), and incubated at 28° C. overnight. Isolates that grew similarlywhen streaked in the presence or absence of antibiotic were classifiedas antibiotic resistant. to test isolates for pigment production, theywere grown on MES minimal medium at 28° C. for seven days and thenscored visually. MES minimal medium contained 9.76 g/L 2-[N-morpholine]ethansulfonic acid (MES), 2 g/L (NH₄)₂ SO₄, 0.2 g/L MgSO₄ ·7 H₂ O,0.25mg/L MnSO₄ ·7 H₂ O, 1.25 g/L K₂ HPO₄ ·3H₂ O, 2 g/L L-glutamic acid,10 mg/L thiamine, 15 g/L agar, 40 mg/L FeCl₃ ·6H₂ O, 6 g/L sucrose and 1mM of the amino acids threonine, serine, leucine, valine, and alanine,and was adjusted to pH 6.1. MES-Thr medium was MES minimal mediumlacking threonine. We characterized the ability of isolates to grow onMES-Thr media by streaking isolates onto MES-Thr plates and incubatingat 28° C. for four days and recording the rate of appearance of coloniesfor each strain. Phages ΦATCC 7064 and ATCC 27877 were obtained from theAmerican Type Culture Collection and were propagated on bacterialstrains ATCC 7064 and ATCC 27877, respectively. Phage Φ63 was propagatedon strain Bt-1, and both Φ63 and Bt-1 were obtained from R. Landen.Sensitivity of isolates to phages Φ63, ΦATCC7064 and ΦATCC27877 wasdetermined by the soft-agar overlay method described above for P7, withplaque formation as the indicator of sensitivity.

Association of zwittermicin A production with P7^(s) and Eh⁺ isolates

It was known that B. cereus strain UW85 produces two antibiotics, thenovel aminopolyol, zwittermicin A, and antibiotic B, that contribute tothe suppression of alfalfa seedling damping-off. UW85 was originallyidentified in a labor-intensive screen for biological control activity.The study conducted above was intended to investigate whethersensitivity to P7 (P7^(s)) and the ability to inhibit E. herbicola (Eh⁺)were phenotypes that could be used to identify zwittermicin A producersand useful biocontrol strains.

4,307 B. cereus and B. thuringiensis isolates were screened for P7^(s)and/or Eh⁺ phenotypes. The isolates were obtained from geographicallydiverse soil samples collected at a total of 16 locations in fivecountries (Table 1 above), from alfalfa and soybean roots, and fromstock culture collections (Table 2 above). the number of P7^(s) orP7^(r) Eh⁺ isolates identified from each source and the number ofisolates tested were tabulated. P7^(s) isolates were identified insamples from 14 of the 16 soils examined as well as from alfalfa andsoybean roots. Of the 87 P7^(s) isolates, all were Eh⁺ except SNY73 andLN100. P7^(r) Eh⁺ isolates were identified from each of the soils aswell as from alfalfa roots. Among all the isolates tested, approximately2% (85/4,307) of the isolates examined were P7^(s) Eh⁺ and 7%(132/1,876) were P7^(r) Eh⁺.

Alfalfa field stand studies

Several of the most promising strains were then used as biocontrolinocula in alfalfa fields grown in Arlington, Hancock, Marshfield andWest Madison, Wis. The results of those field trials are set forth inTables 3, 4, 5 and 6 below.

From that data, it was determined that strain AS4-12 had, overall, thebest field performance in enhancing the stand of alfalfa fields undernormal cultivation conditions in the upper midwestern U.S.

                                      TABLE 3                                     __________________________________________________________________________    Biocontrol by Bacillus cereus strains on alfalfa at Arlington, WI             Emergence counts.sup.a    Forage yield.sup.b  Stand                                 07 DAP.sup.d                                                                       14 DAP                                                                             21 DAP                                                                             28 DAP                                                                             Harvest                                                                            Harvest                                                                            Harvest                                                                            Harvest                                                                            count.sup.c                     Treatment                                                                           5-26-93                                                                            6-02-93                                                                            6-09-93                                                                            6-16-93                                                                            7-29-93                                                                            9-16-93                                                                            6-03-94                                                                            7-18-94                                                                            8-02-94                         __________________________________________________________________________    Untreated                                                                           35.8 61.8 52.2 44.8 182.8                                                                              281.4                                                                              985.4                                                                              465.0                                                                              28.2                            Apron 39.2 56.4 46.2 42.4 260.6                                                                              346.2                                                                              962.0                                                                              470.8                                                                              25.6                            UW85  41.4 65.0 51.2 44.2 232.2                                                                              366.6                                                                              1117.2                                                                             460.0                                                                              29.0                            AP 17-5                                                                             48.6 71.6 61.8 54.2 237.2                                                                              324.6                                                                              934.0                                                                              466.4                                                                              --                              AP 2-1                                                                              36.6 58.6 44.6 39.0 206.0                                                                              300.8                                                                              920.4                                                                              459.4                                                                              --                              AS 4-12                                                                             55.0 89.4 73.6 56.8 154.4                                                                              303.4                                                                              1005.0                                                                             443.8                                                                              34.2                            AS 23-15                                                                            32.6 58.4 43.8 38.6 241.4                                                                              323.8                                                                              1057.8                                                                             453.8                                                                              --                              HP 20-2                                                                             32.6 53.4 36.0 33.8 208.8                                                                              302.2                                                                              1140.6                                                                             480.2                                                                              --                              HP 24-2                                                                             43.4 75.4 68.8 59.2 305.4                                                                              330.2                                                                              967.6                                                                              462.0                                                                              --                              HS 1-3                                                                              42.2 59.8 48.2 42.0 239.6                                                                              337.0                                                                              1007.8                                                                             481.4                                                                              --                              HS 18-4                                                                             36.2 65.6 47.2 48.6 220.6                                                                              296.4                                                                              1070.6                                                                             427.6                                                                              --                              LP 34-16                                                                            40.4 65.2 51.2 43.8 310.8                                                                              354.0                                                                              1022.0                                                                             448.2                                                                              --                              LP 38-2                                                                             38.2 64.0 49.2 42.8 348.8                                                                              433.6                                                                              1011.6                                                                             504.8                                                                              --                              LP 4-13                                                                             40.8 63.2 46.8 45.8 285.4                                                                              367.8                                                                              1091.4                                                                             447.2                                                                              --                              LP 15-3                                                                             32.2 59.8 50.2 39.2 228.0                                                                              381.8                                                                              1201.0                                                                             480.2                                                                              --                              MS 1-9                                                                              37.2 60.8 45.0 39.6 274.4                                                                              379.0                                                                              1105.4                                                                             442.4                                                                              --                              MS 19-4                                                                             26.8 52.4 44.4 37.0 280.6                                                                              316.8                                                                              938.6                                                                              500.4                                                                              --                              WP 27-3                                                                             42.2 63.0 52.6 43.0 260.4                                                                              324.6                                                                              957.6                                                                              387.8                                                                              --                              WP 36-16                                                                            42.2 63.2 52.6 43.0 204.4                                                                              297.6                                                                              804.4                                                                              345.4                                                                              --                              WS 12-3                                                                             48.2 74.6 61.8 52.6 246.2                                                                              303.8                                                                              1073.6                                                                             483.4                                                                              --                              WS 12-4                                                                             46.0 67.8 52.8 40.6 255.6                                                                              373.0                                                                              1066.0                                                                             440.8                                                                              --                              Pr> F 0.0415                                                                             0.0019                                                                             0.0005                                                                             0.0436                                                                             0.9441                                                                             0.9719                                                                             0.7041                                                                             0.7891                                                                             0.2299                          LSD (0.05)                                                                          13.7 14.7 14.6 13.9 174.1                                                                              174.1                                                                              281.3                                                                              118.6                                                                              8.6                             C.V.  27.3 18.2 22.6 25.0 56.0 36.9 21.9 20.7 21.4                            __________________________________________________________________________     .sup.a Emergence counts: number of seedlings per 1 m row                      .sup.b Forage yield: forage fresh weight per 1 m row                          .sup.c Stand count: number of plants per 1 m row                              .sup.d DAP = days after planting                                              Field number: 248                                                             Soil type: Plano silt loam                                                    Experimental design: Randomized Complete Block with 5 replications            Cultivar: Iroquois                                                            Planting date: 509-93                                                         Planting rate: 20 lbs per acre (assuming 6 inch rows)                         Fungicide rate: 4 oz per 100 lbs seed                                         Bacteria rate: 12 fluid oz per 60 lbs seed (10.sup.8 cells per ml of          culture)                                                                 

                                      TABLE 4                                     __________________________________________________________________________    Biocontrol by Bacillus cereus strains on alfalfa at Hancock, WI               Emergence counts.sup.a    Forage yield.sup.b  Stand                                 07 DAP.sup.d                                                                       14 DAP                                                                             21 DAP                                                                             28 DAP                                                                             Harvest                                                                            Harvest                                                                            Harvest                                                                            Harvest                                                                            count.sup.c                     Treatment                                                                           6-02-93                                                                            6-09-93                                                                            6-16-93                                                                            6-23-93                                                                            8-05-93                                                                            9-23-93                                                                            6-06-94                                                                            7-22-94                                                                            8-04-94                         __________________________________________________________________________    Untreated                                                                           53.6 57.4 45.6 41.4 68.0 151.4                                                                              671.2                                                                              478.6                                                                              39.2                            Apron 75.4 77.2 59.4 59.2 61.6 137.0                                                                              596.2                                                                              431.8                                                                              52.8                            UW85  55.6 64.0 50.6 48.2 79.8 169.8                                                                              762.4                                                                              530.6                                                                              45.4                            AP 17-5                                                                             44.0 44.0 37.2 33.8 51.4 153.2                                                                              642.0                                                                              478.2                                                                              --                              AP 2-1                                                                              45.0 42.8 40.4 37.8 52.8 159.8                                                                              624.0                                                                              368.4                                                                              --                              AS 4-12                                                                             92.6 81.2 73.0 64.8 71.0 154.2                                                                              634.8                                                                              449.6                                                                              55.8                            AS 23-15                                                                            54.0 47.0 46.0 40.8 61.6 178.0                                                                              735.0                                                                              481.8                                                                              --                              HP 20-2                                                                             45.2 40.4 37.0 34.0 51.8 129.4                                                                              679.2                                                                              438.2                                                                              --                              HP 24-2                                                                             53.4 54.2 46.6 43.0 58.0 155.2                                                                              717.2                                                                              551.4                                                                              --                              HS 1-3                                                                              53.2 47.2 41.2 36.8 60.4 147.6                                                                              667.2                                                                              431.4                                                                              --                              HS 18-4                                                                             57.2 61.6 50.6 47.2 76.0 160.8                                                                              653.8                                                                              453.2                                                                              --                              LP 34-16                                                                            55.6 55.0 53.0 46.0 70.2 152.2                                                                              652.8                                                                              426.6                                                                              --                              LP 38-2                                                                             50.6 51.6 43.8 39.0 46.8 118.8                                                                              600.8                                                                              422.6                                                                              --                              LP 4-13                                                                             63.6 57.0 55.2 51.0 73.6 160.6                                                                              684.0                                                                              405.4                                                                              --                              LP 15-3                                                                             52.8 52.2 44.0 42.2 66.8 166.2                                                                              654.4                                                                              453.2                                                                              --                              MS 1-9                                                                              61.0 53.0 52.0 47.8 74.2 153.2                                                                              703.4                                                                              475.4                                                                              --                              MS 19-4                                                                             47.4 53.6 45.0 43.0 61.6 140.0                                                                              649.0                                                                              507.8                                                                              --                              WP 27-3                                                                             45.0 49.2 45.6 42.4 67.0 145.4                                                                              669.2                                                                              469.6                                                                              --                              WP 36-16                                                                            60.2 55.4 49.8 44.6 61.2 154.8                                                                              650.8                                                                              462.6                                                                              --                              WS 12 3                                                                             58.4 51.2 48.4 45.0 61.8 163.6                                                                              643.8                                                                              475.4                                                                              --                              WS 12-4                                                                             55.6 50.6 46.4 40.2 46.8 128.0                                                                              590.0                                                                              392.6                                                                              --                              Pr> F 0.0001                                                                             0.0002                                                                             0.0005                                                                             0.0003                                                                             0.0231                                                                             0.6947                                                                             0.7181                                                                             0.2460                                                                             0.0155                          LSD (0.05)                                                                          13.0 16.2 13.3 12.3 19.3 45.2 137.6                                                                              109.8                                                                              10.1                            C.V.  18.3 23.5 21.9 22.1 24.4 23.7 16.5 19.1 15.2                            __________________________________________________________________________     .sup.a Emergence counts: number of seedlings per 1 m row                      .sup.b Forage yield: forage fresh weight per 1 m row                          .sup.c Stand count: number of plants per 1 m row                              .sup.d DAP = days after planting                                              Field number: V21                                                             Soil type: Plainfield loamy sand                                              Experimental design: Randomized Complete Block with 5 replications            Cultivar: Iroquois                                                            Planting date: 526-93                                                         Planting rate: 20 lbs per acre (assuming 6 inch rows)                         Fungicide rate: 4 oz per 100 lbs seed                                         Bacteria rate: 12 fluid oz per 60 lbs seed (10.sup.8 cells per ml of          culture)                                                                 

                                      TABLE 5                                     __________________________________________________________________________    Biocontrol by Bacillus cereus strains on alfalfa at Marshfield, WI            Emergence counts.sup.a    Forage yield.sup.b  Stand                                 07 DAP.sup.d                                                                       14 DAP                                                                             21 DAP                                                                             28 DAP                                                                             Harvest   Harvest                                                                            Harvest                                                                            count.sup.c                     Treatment                                                                           7-06-93                                                                            7-13-93                                                                            7-20-93                                                                            7-27-93                                                                            8-25-93                                                                            Harvest                                                                            6-07-94                                                                            7-21-94                                                                            8-03-94                         __________________________________________________________________________    Untreated                                                                           32.0 28.2 26.4 28.8 191.0                                                                              --   731.4                                                                              525.8                                                                              27.8                            Apron 78.2 66.2 64.2 60.2 260.8                                                                              --   826.4                                                                              631.8                                                                              52.6                            UW85  41.2 32.4 33.2 33.6 235.8                                                                              --   919.0                                                                              557.0                                                                              33.6                            AP 17-5                                                                             35.4 28.8 27.6 29.6 211.4                                                                              --   785.6                                                                              580.0                                                                              --                              AP 2-1                                                                              33.4 29.8 29.2 30.0 221.2                                                                              --   872.2                                                                              597.8                                                                              --                              AS 4-12                                                                             70.8 65.2 62.2 58.4 288.2                                                                              --   974.4                                                                              677.6                                                                              54.0                            AS 23-15                                                                            36.0 31.0 30.2 28.4 217.8                                                                              --   847.4                                                                              451.8                                                                              --                              HP 20-2                                                                             36.6 31.4 31.2 32.2 219.2                                                                              --   843.4                                                                              540.2                                                                              --                              HP 24-2                                                                             28.2 24.8 23.0 24.8 215.2                                                                              --   816.8                                                                              550.2                                                                              --                              HS 1-3                                                                              45.8 38.4 36.6 36.6 222.8                                                                              --   825.2                                                                              556.0                                                                              --                              HS 18-4                                                                             40.8 34.4 29.0 32.4 212.2                                                                              --   880.0                                                                              582.6                                                                              --                              LP 34-16                                                                            36.8 36.6 34.6 33.0 246.2                                                                              --   883.8                                                                              577.0                                                                              --                              LP 38-2                                                                             36.8 34.8 34.2 33.8 229.0                                                                              --   840.6                                                                              525.0                                                                              --                              LP 4-13                                                                             36.0 30.0 28.8 29.4 218.0                                                                              --   775.0                                                                              533.0                                                                              --                              LP 15-3                                                                             36.2 31.2 27.2 30.6 202.6                                                                              --   810.0                                                                              550.8                                                                              --                              MS 1-9                                                                              42.0 37.8 36.6 36.2 250.2                                                                              --   948.8                                                                              602.4                                                                              --                              MS 19-4                                                                             36.4 34.4 33.4 33.6 233.0                                                                              --   828.6                                                                              553.0                                                                              --                              WP 27-3                                                                             30.4 26.4 24.6 26.8 218.8                                                                              --   727.4                                                                              562.4                                                                              --                              WP 36-16                                                                            35.4 33.0 30.4 30.2 226.2                                                                              --   837.2                                                                              569.2                                                                              --                              WS 12-3                                                                             29.2 23.0 23.8 23.0 186.2                                                                              --   780.0                                                                              478.0                                                                              --                              WS 12-4                                                                             39.6 35.0 36.4 33.2 227.0                                                                              --   831.8                                                                              520.8                                                                              --                              Pr> F 0.0001                                                                             0.0001                                                                             0.0001                                                                             0.0001                                                                             0.4338                                                                             --   0.2815                                                                             0.0642                                                                             0.0001                          LSD (0.05)                                                                          11.5 10.5 10.8 9.9  63.0 --   160.8                                                                              106.9                                                                              8.2                             C.V.  23.0 23.8 25.7 23.4 22.2 --   15.3 15.2 14.2                            __________________________________________________________________________     .sup.a Emergence counts: number of seedlings per 1 m row                      .sup.b Forage yield: forage fresh weight per 1 m row                          .sup.c Stand count: number of plants per 1 m row                              .sup.d DAP = days after planting                                              Field number: W9                                                              Soil type: Withee silt loam                                                   Experimental design: Randomized Complete Block with 5 replications            Cultivar: Iroquois                                                            Planting date: 629-93                                                         Planting rate: 20 lbs per acre (assuming 6 inch rows)                         Fungicide rate: 4 oz per 100 lbs seed                                         Bacteria rate: 12 fluid oz per 60 lbs seed (10.sup.8 cells per ml of          culture)                                                                 

                                      TABLE 6                                     __________________________________________________________________________    Biocontrol by Bacillus cereus strains on alfalfa at West Madison, WI          Emergence counts.sup.a    Forage yield.sup.b  Stand                                 07 DAP.sup.d                                                                       14 DAP                                                                             21 DAP                                                                             28 DAP                                                                             Harvest                                                                            Harvest                                                                            Harvest                                                                            Harvest                                                                            count.sup.c                     Treatment                                                                           5-28-93                                                                            6-04-93                                                                            6-11-93                                                                            6-18-93                                                                            8-04-93                                                                            9-24-93                                                                            6-01-94                                                                            7-18-94                                                                            8-01-94                         __________________________________________________________________________    Untreated                                                                           68.2 72.6 64.0 60.6 40.4 252.8                                                                              935.2                                                                              522.4                                                                              44.8                            Apron 80.0 86.6 63.0 69.2 24.2 156.0                                                                              868.2                                                                              440.0                                                                              44.6                            UW85  77.4 86.2 69.0 66.2 51.6 241.6                                                                              924.4                                                                              502.8                                                                              44.2                            AP 17-5                                                                             64.8 67.8 60.8 54.4 31.4 214.6                                                                              888.0                                                                              494.8                                                                              --                              AP 2-1                                                                              62.6 71.0 62.8 62.4 33.2 222.8                                                                              940.4                                                                              520.8                                                                              --                              AS 4-12                                                                             89.6 96.4 87.0 80.2 45.0 248.4                                                                              936.2                                                                              517.0                                                                              51.4                            AS 23-15                                                                            70.4 80.8 64.0 57.8 20.6 221.4                                                                              865.4                                                                              498.6                                                                              --                              HP 20-2                                                                             69.4 82.0 64.6 58.8 36.0 240.0                                                                              933.4                                                                              530.2                                                                              --                              HP 24-2                                                                             66.6 77.8 68.6 63.2 41.8 223.0                                                                              919.0                                                                              440.2                                                                              --                              HS 1-3                                                                              70.4 70.6 62.2 56.2 38.4 246.0                                                                              931.0                                                                              499.6                                                                              --                              HS 18-4                                                                             68.6 75.2 66.2 64.6 40.8 230.2                                                                              910.8                                                                              466.0                                                                              --                              LP 34-16                                                                            75.6 85.4 61.0 67.0 23.4 218.0                                                                              884.6                                                                              498.6                                                                              --                              LP 38-2                                                                             76.2 83.2 61.2 58.4 22.2 184.6                                                                              911.6                                                                              517.4                                                                              --                              LP 4-13                                                                             75.4 76.2 58.6 56.4 29.8 196.6                                                                              914.6                                                                              463.2                                                                              --                              LP 15-3                                                                             71.6 74.2 63.2 57.4 28.2 229.2                                                                              929.2                                                                              465.4                                                                              --                              MS 1-9                                                                              65.0 82.2 65.0 62.6 37.4 219.6                                                                              891.4                                                                              474.8                                                                              --                              MS 19-4                                                                             60.0 69.6 58.6 49.0 37.8 238.2                                                                              929.0                                                                              419.4                                                                              --                              WP 27-3                                                                             80.0 84.8 65.0 56.8 39.2 230.8                                                                              826.0                                                                              544.6                                                                              --                              WP 36-16                                                                            71.2 74.8 60.6 56.4 48.6 227.0                                                                              939.8                                                                              519.2                                                                              --                              WS 12-3                                                                             61.0 64.4 54.2 51.6 25.4 228.4                                                                              940.6                                                                              498.4                                                                              --                              WS 12-4                                                                             68.8 75.4 63.4 55.0 40.8 257.8                                                                              921.0                                                                              501.4                                                                              --                              Pr> F 0.0011                                                                             0.0231                                                                             0.3987                                                                             0.0470                                                                             0.2839                                                                             0.9080                                                                             0.6085                                                                             0.6016                                                                             0.4005                          LSD (0.05)                                                                          12.3 15.7 17.0 14.7 22.7 85.1 91.0 98.0 10.3                            C.V.  13.8 16.0 21.2 19.4 51.5 30.2 7.9  15.8 16.1                            __________________________________________________________________________     .sup.a Emergence counts: number of seedlings per 1 m row                      .sup.b Forage yield: forage fresh weight per 1 m row                          .sup.c Stand count: number of plants per 1 m row                              .sup.d DAP = days after planting                                              Field number: P11                                                             Soil type: Plano silt loam                                                    Experimental design: Randomized Complete Block with 5 replications            Cultivar: Iroquois                                                            Planting date: 521-93                                                         Planting rate: 20 lbs per acre (assuming 6 inch rows)                         Fungicide rate: 4 oz per 100 lbs seed                                         Bacteria rate: 12 fluid oz per 60 lbs seed (10.sup.8 cells per ml of          culture)                                                                 

What is claimed is:
 1. A biologically pure culture of Bacillus cereuswhich is AS4-12, ATCC
 55609. 2. A biologically pure culture of a mutantstrain of Bacillus cereus strain AS4-12, ATCC 55609 which retain theabilities to produce zwittermicin A and to protect alfalfa plants fromdamping off disease under field conditions.
 3. An inoculum forapplication to alfalfa comprising a carrier and an effective quantity ofa bacteria selected from the group consisting of Bacillus cereus AS4-12,ATCC 55609, mutants of Bacillus cereus AS4-12, ATCC 55609, which retainthe capabilities to produce zwittermicin A and to protect alfalfa plantsfrom damping off disease, and mixtures thereof.
 4. A method forprotecting plants in a growing medium from damping off diseasecomprising the steps of placing in the vicinity of the plant to beprotected an effective quantity of a bacteria selected from the groupconsisting of Bacillus cereus AS4-12, ATCC 55609 and mutants thereofwhich retain the capabilities of producing zwittermicin A and protectingalfalfa plants from damping off disease.